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Abstract

CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K cDNA transfection abolished CD9 mediated pro-migratory effects. Consistent with these observations, CD9 expression in CHO cells and in the rat aortic smooth muscle (RASM) cells induced enhanced phosphorylation of PI-3K substrate, Akt. In CHO cells, CD9 expression also enhanced protein levels and tyrosine phosphorylation of the adaptor protein p130Cas. However, no significant changes in the CD9 enhanced migration were observed in CHO cells upon down regulation of p130Cas CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K cDNA transfection abolished CD9 mediated pro-migratory effects. Consistent with these observations, CD9 expression in CHO cells and in the rat aortic smooth muscle (RASM) cells induced enhanced phosphorylation of PI-3K substrate, Akt. In CHO cells, CD9 expression also enhanced protein levels and tyrosine phosphorylation of the adaptor protein p130Cas. However, no significant changes in the CD9 enhanced migration were observed in CHO cells upon down regulation of p130Cas by siRNA transfection suggesting that p130Cas dependent pathways are not mandatory for CD9 mediated motility. To further understand the mechanisms by which CD9 regulates cell migration, we studied the relative contribution of the fibronectin (FN) receptor integrin, alpha-5-beta-1 in CD9 mediated cell motility. Our findings show that CD9 is in molecular complex with alpha-5-beta-1 and that CD9 promoted migration can be completely blocked by an alpha-5-beta-1 function blocking antibody. Further studies revealed that CD9 expression may stabilize the active conformer of the beta-1 integrin. Taken togeather, our study demonstrates key molecular mechanisms governing CD9 mediated haptotactic cell motility to FN in CHO cells. Our findings indicate that CD9 in concert with integrin alpha-5-beta-1 requires activation of the PI-3K pathway leading to enhanced haptotactic cell migration on FN.